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Aneuploidy Underlies a Multicellular Phenotypic Switch

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Aneuploidy Underlies a Multicellular Phenotypic Switch Aneuploidy underlies a multicellular phenotypic switch Zhihao Tana,b,1, Michelle Haysa,1, Gareth A. Cromiea, Eric W. Jefferya, Adrian C. Scotta, Vida Ahyongc, Amy Sirra, Alexander Skupina,d, and Aimée M. Dudleya,b,2 aInstitute for Systems Biology, Seattle, WA 98109; bMolecular and Cellular Biology Program, University of Washington, Seattle, WA 98105; cTetrad Program, University of California, San Francisco, CA 94122; and dLuxembourg Center for Systems Biomedicine, University of Luxembourg, L-4362 Esch-sur-Alzette, Luxembourg Edited by Angelika Amon, Massachusetts Institute of Technology, Cambridge, MA, and approved May 31, 2013 (received for review January 25, 2013) Although microorganisms are traditionally used to investigate and wrinkled colonies: state changes that are associated with unicellular processes, the yeast Saccharomyces cerevisiae has the biochemically distinct capsular polysaccharides (18), changes in ability to form colonies with highly complex, multicellular struc- karyotype (18), and differential fungal burden in infected host tures. Colonies with the “fluffy” morphology have properties rem- organs (19). Because these different states may exhibit differ- iniscent of bacterial biofilms and are easily distinguished from the ential virulence, cell surface properties, or resistance to host im- “smooth” colonies typically formed by laboratory strains. We have mune defenses (2, 3), understanding the mechanisms underlying identified strains that are able to reversibly toggle between the phenotypic switching could prove essential for the development of fluffy and smooth colony-forming states. Using a combination of effective therapeutics against microbial pathogens. flow cytometry and high-throughput restriction-site associated Some strains of S. cerevisiae also exhibit a striking phenotypic DNA tag sequencing, we show that this switch is correlated with switch. Although most laboratory strains produce relatively un- a change in chromosomal copy number. Furthermore, the gain of structured “smooth” colonies, some natural isolates of S. cer- a single chromosome is sufficient to switch a strain from the fluffy evisiae produce colonies with complex multicellular features, to the smooth state, and its subsequent loss to revert the strain such as folds, crevices, and channels that form as the colony back to the fluffy state. Because copy number imbalance of six of grows on solid media (20, 21). These “fluffy” colonies possess the 16 S. cerevisiae chromosomes and even a single gene can mod- properties similar to those of microbial biofilms, including the ulate the switch, our results support the hypothesis that the state secretion and maintenance of an extracellular matrix (21–23), switch is produced by dosage-sensitive genes, rather than a general localized expression of drug efflux pumps (23), increased ad- response to altered DNA content. These findings add a complex, herence (24), and the use of cell–cell communication (25). Fluffy multicellular phenotype to the list of molecular and cellular traits colony formation requires the function and coordination of nu- known to be altered by aneuploidy and suggest that chromosome merous pathways that underlie the trait, and the deletion of key missegregation can provide a quick, heritable, and reversible mech- factors produces a smooth colony phenotype (26–28). Fluffy anism by which organisms can toggle between phenotypes. colonies may benefit from both rapid acquisition of larger ter- ritories as well as an increased capacity for nutrient and water colony morphology | copy number variation | phenotypic switching | transport (21). Interestingly, some fluffy strains can switch to the bet-hedging smooth morphology at a rate significantly higher than that expected for spontaneous mutations (21, 29). henotypic switching allows cells or organisms to reversibly Results Ptoggle between multiple, heritable states (1–3). These phe- notypic states can confer distinct properties to cells that may be Bidirectional Toggling Between Phenotypic States. While studying advantageous in some environments, e.g., increased resistance to colony morphology in the haploid progeny of a cross between a Japanese sake strain and an Ethiopian white tecc strain (SI heat (4), faster proliferation rates in the presence of excess − Appendix 3 , Table S1), we observed a high frequency (10 )ofcol- GENETICS nutrients (5), or altered interaction with host immune systems fl (6). Although some phenotypic switches can be induced by onies that switched from a uffy to a smooth morphology. The a change in environment (7), others may arise stochastically, smooth state of these isolates was clonally heritable and stable possibly as a bet-hedging strategy that increases the probability through multiple cell divisions, as evidenced by the formation of smooth colonies from single cells isolated from 4-d-old smooth that a subpopulation of genetically isogenic cells survive a wider colonies (Fig. 1A). However, when grown for longer periods of range of environmental perturbations (4, 8, 9). – “ ” The ability to switch phenotypes could prove especially useful time (10 16 d), blebs appeared on the colony surface of otherwise smooth colonies (Fig. 1B). We hypothesized that these for microorganisms that are unable to physically relocate to patches of cells had adopted characteristics distinct from the an environment of choice and need to weather environmental neighboring cells, and indeed, a portion of single cells isolated fluctuations. Indeed, microbes are known to adopt multiple from these patches had regained the ability to form fluffy colonies phenotypic states, and though the nature of these states can vary widely, they are all characterized by rates of switching too high to be that of spontaneous mutation (2, 3). Several interesting Author contributions: Z.T., M.H., G.A.C., E.W.J., A. Skupin, and A.M.D. designed research; examples have been discovered in opportunistic fungal patho- Z.T., M.H., E.W.J., A.C.S., V.A., and A. Sirr performed research; G.A.C., A.C.S., V.A., and A. Sirr gens. Perhaps the best characterized state switch in fungi is the contributed new reagents/analytic tools; Z.T., M.H., G.A.C., A.C.S., and A. Skupin analyzed white-opaque switch of the Candida albicans (10), which is reg- data; and Z.T., M.H., and A.M.D. wrote the paper. ulated by transcriptional feedback loops (11) and is associated The authors declare no conflict of interest. with differential virulence (12), host colonization (13) and mat- This article is a PNAS Direct Submission. ing competency (14). C. albicans can also differentiate a sub- Freely available online through the PNAS open access option. “ ” population of persister cells that, like their bacterial counterparts, Data deposition: Raw restriction-site associated DNA tag sequencing reads of strains for exhibit increased resistance to antimicrobial drugs (15). Candida which ploidy was analyzed are deposited in the National Center for Biotechnology In- glabrata exhibits at least two spontaneous and reversible phe- formation Sequence Read Archive, www.ncbi.nlm.nih.gov/sra/ (accession no. ERP002462). notypic switches: a “core switching system” and an “irregular 1Z.T. and M.H. contributed equally to this work. wrinkle switching system” (16) which exhibit differential tissue 2To whom correspondence should be addressed. E-mail: [email protected]. colonization and clearing rates in vivo (17). Cryptococcus This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. neoformans switches between smooth, mucoid, pseudohyphal, 1073/pnas.1301047110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1301047110 PNAS | July 23, 2013 | vol. 110 | no. 30 | 12367–12372 Downloaded by guest on September 28, 2021 based on ∼3,000 markers across the genome. Together with DNA content estimates determined by flow cytometry (SI Appendix, Fig. S2), RAD-seq showed that although the original fluffy strain (F45) was euploid, transition to the smooth state (F45 Smooth) was accompanied by the gain of an extra copy of chromosome XVI (Fig. 2A). Remarkably, the additional copy of chromosome XVI was lost when the strain transitioned back to the fluffy state (Fig. 2A,F45“2nd gen”), exhibiting a morphology indistinguishable from that of the original strain. To test whether this phenomenon was specific to F45, we chose a strain (YO880) from an independent genetic background that also harbored an extra copy of chromosome XVI. YO880 is a haploid strain derived from a cross between strains originating from “Evolution Canyon” in Israel and an oak tree in North America (SI Appendix, Table S1). Similar to F45, the version of YO880 containing an extra copy of chromosome XVI was smooth (Fig. 2B), but upon extended growth produced patches of fluffy colony producing cells. RAD-seq confirmed that these fluffy iso- lates had also lost the extra copy of chromosome XVI (Fig. 2B). Thus, in two independent genetic backgrounds, smooth and fluffy colony morphologies were correlated with chromosome XVI copy number. To determine whether chromosome XVI disomy was sufficient for the phenotypic switch, we engineered the original strain (F45) to enrich for chromosome XVI disomes, independent of their phenotype (Fig. 3). This system (32) modifies the centro- mere of a target chromosome so that it can be temporarily inactivated (by exposure to galactose), thereby increasing the frequency of chromosome-specific nondisjunction (33). Galac- tose-induced nondisjunction events are selected using a marker that can exist in one of two states, an intact HIS3 or an intact URA3 (32). Only cells that have obtained two copies of the target chromosome can excise HIS3 from one copy and maintain URA3 on the other, allowing them to grow on the selective medium (SC–His–Ura). In our strain background, ∼103 out of 107 ga- + + lactose-induced cells produced His Ura colonies. Because colony morphology was difficult to score on the selective medium Fig. 1. Toggling between the fluffy and smooth states. (A) Morphology (SI Appendix, Fig. S3), we randomly chose 48 colonies and development of a colony growing from a single cell of the F45 fluffy strain assayed their morphology on rich medium (YPD) (SI Appendix, and a smooth variant (F45 Smooth).
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